Enteric viruses are mostly found in different aquatic reservoirs, including ground water, still water, streams, rivers, and r water. They are associated with health risks and are responsible for infections in humans. Enteric viruses commonly cause gastroenteritis and are mostly transmitted through the fecal-oral route.
Other waterborne diseases cause acute hepatitis by hepatitis A virus and hepatitis E viruses, with significant associated mortality especially regarding hepatitis E in pregnant women. Actual protocols for monitoring water quality proposed for humanitarian contexts have been designed to keep bacteria such as E.Coli as an indicator of fecal contamination. But it's already known that there is no correlation with other microorganisms such as viruses that are more resistant to many activation processes.
Until now, detection of viruses in humanitarian crisis settings have been performed by adapting existing protocols to the conditions of those contexts, and shipping samples to reference laboratories for molecular detection of viruses. There is a need of methods to detect viral pathogens and viral indicators in the point of views to reduce public health risks, preventing outbreaks and increasing the efficiency of humanitarian interventions. We propose a method for the detection of viruses in 10 liter water samples which is divided into three steps:Viral concentration, nucleic acid extraction, and viral detection.
All these steps have been adapted from methods currently in use in our laboratory, and can be performed in the point of views by using portable reagents and equipment, producing the dependents of power supply and freezers. Also, a preservative solution has been added to the method to warrant the stability of nucleic acids for shipment to reference laboratories if needed. The method we propose is called VirWaTest and has been developed with the collaboration of GenIUL and Intermon Oxfam.
Before starting, the number of samples to be tested should be considered for preparation of reagents and material. It is recommended to collect two replicates of each water sample in order to obtain representative results. Several samples can be tested at the same time if the appropriate amount of material is available.
Reagents and materials should be prepared and packed before moved or shipped to the place where the method is needed. The list of materials and small equipment that should be packed is described in table one. Prepare a bacteriophage MS2 stock to be added to each water sample as a process control by following the corresponding ISO method.
Then distribute 10 microliters of a suspension containing 10 to 10 platforming units pre-made into 10 mL tubes and let the water dry. One tube per water sample will be needed. Additionally, prepare one tube containing 10 mLs of sterile distilled water per collected sample.
For the pre-flocculated skimmed milk solution, prepare a tube containing the skimmed milk, a zipper plastic bag containing the sea salt, and a plastic bottle containing the distilled water. For pH adjustment, prepare a tube containing the citric acid, a tube containing the sodium hydroxide, and two plastic containers with 25 and 50 mLs of distilled water. For each water sample to be tested, prepare a conditioning sachet by adding the sea salts and the citric acid to a zipper plastic bag, and a preserving solution by adding the lysis buffer, and nucleic acid preservative, to a 5 mL tube.
Finally, prepare four neutralizing sachets, adding power detergent to four zipper plastic bags. For each water sample to be extracted, prepare one five mL tube containing magnetic particles and ethanol, and three 2 mL tubes containing 500, 600, and 200 microliters of washing buffers one, two, and three, respectively, and one tube containing elution buffer. Two different nucleic acid extractions may be analyzed simultaneously in each PCR assay if a Newell thermocycler is used.
For all PCR assays, prepare molecular biology water. Prepare mixes containing two primers and one proof specific for each target virus according to the volumes and concentrations described in the protocol. For each virus, add the indicated volumes into tubes one to eight of a tubes tray.
Cut the strip, dividing it into two strips. Tubes one to five and tubes six to eight. For each virus, air dry DNA suspensions containing 10 to 5, 10 to 7, and 10 to 9 and known copies pre-made into tubes six, seven, and eight.
This concentration protocol is based on the principle of organic flocculation by which the low pH and high-conductivity condition drives the skim milk proteins to organize into flocs the viruses absorb to. Once the flocs are settled, they can be easily be collected. Collect a 10 liter water sample in a flat-bottom bucket provided with a lid.
If collecting from a pipe, let the water run for a few seconds before collecting the water. It's important to use clear buckets to visualize pellets. Look for a flat space in a fresh place far from direct sunlight Put a magnetic stirrer connected to a battery under the bucket support and place the bucket containing the water sample on each support.
Reconsider the citric acid and the sodium hydroxide and shake until they are completely dissolved. Pour 500 mLs of distilled water into a stand-up bag and place the bag on a magnetic stirrer. Add the contents of one sea salt sachet and the contents of one skim milk tube and stir for five minutes at medium speed.
Adjust the pH of the flocculated skim milk using citric acid and sodium hydroxide to a pH between 3.4 and 3.6. Turn off the magnetic stirrer and make sure the flocs are visible. A flashlight may help in visualizing the flocs.
Drop a stirring magnet into the water. Set the magnetic stirrer at maximum speed and turn it on. Add 10 mLs of distilled water to a process-controlled tube.
Invert it a few times and pour the solution into the water. Pour the contents of one sample conditioning sachet into the water and stir for five minutes. Make sure the pH of the water sample, which should be between 3.4 and 3.6.
Adjust the pH if required. Add 100 mLs of pre-flocculated skim milk solution, seal the bucket with the lid and leave the water stirring for eight hours at minimum speed. After eight hours, turn off the magnetic stirrer and allow the flocs to settle for at least five extra hours.
With a system based on the use of a pipette controller, a plastic tube, and a couple of pipettes, discard the supernatant, taking care not to disturb the flocs on the bucket bottom. When the water level is about to reach the stirring magnet, squeeze the plastic tube to stop the water flow and move away the pipette from the bucket. Shake the bucket to resuspend the flocs and pour them into a stand-up plastic bag.
Allow the flocs to settle for one extra hour. Carefully aspirate the water supernatant using a pipette avoiding to disturb the flocs. Aspirate the flocs using a pipette and transfer them into a conical tube.
Note down the final volume of sample concentrate and transfer 1 mL to a tube containing the preservative solution. The suspension can be either used to perform the nucleic acid extraction in the field, or ship to a reference laboratory for further analysis. The water discarded while collecting the flocs must be treated before disposal.
Add the contents of a neutralizing agent sachet to the bucket containing the discarded water. Mix the water and leave it still for thirty minutes. Then dispose of the treated water as general waste.
The magnetic particles can be transferred from one tube to another using a magnetic pipette. Get used to the operation of a magnetic pipette before performing the extraction. Arrange the tubes containing the reagent required for extraction in a rack.
That is, from left to right:binding buffer, washing buffers, and elution buffer. Transfer 1 mL of sample concentrate to the tube containing the binding buffer using a disposable plastic pipette. Invert the tube ten times to optimize the solution.
Incubate the solution at room temperature for ten minutes while continuously mixing either using a solo orbital or manually. Collect the magnetic particles using a magnetic pipette and a clean tip that can be reused for the three washing buffers and release the magnetic particles into the tube containing washing buffer one. Wash them by gently shaking the solution with the tip for thirty seconds and collect them.
Repeat this washing procedure for washing buffers two and three. Collect the magnetic particles in washing buffer three and release them into the tube containing the elution buffer. Then discard the tip.
Incubate the solution at room temperature for five minutes while continuously mixing using the solo orbital. Replace the tip on the magnetic pipette by any one and collect all the magnetic particles from the elution buffer. Discard the tip with the particles attached.
The nucleic acid remains in the elution buffer, which should be stored for further analysis or shipment. Use a five-tube and a three-tube strip prepared to detect human adenoviruses. Add water, PCR mix, and nucleic acid extration to tubes one through five according to the protocol.
Afterwards, add water and PCR mix to tubes six through eight. Put both strips in the thermocycler and select the appropriate run. Once the run is finished, collect the results and analyze obtained data.
Only when viral tests have resulted in negative, MS2 detection should be performed. Use tube strips prepared for MS2 detection and select the appropriate thermal profile for the PCR. The VirWaTest magnetic nucleic acid extraction was compared to QIAgen viral RNA, mimicked it by testing water samples spiked with human adenoviruses and MS2.
Reduction test p-value shows significantly higher viral recoveries by using VirWaTest than QiAgen as an extraction method for both viruses tested. The developed method for viral concentration was tested in the field with the collaboration of Oxfam wash teams located in Bangui, Central African Republic and in Pedernales, Ecuador, who collected water samples, concentrated them by applying the developed concentration method, and sent them to the laboratory in Barcelona for nucleic acid extraction and viral quantification. In Ecuador, human adenoviruses were detected in all samples tested, whereas one out of five samples collected and concentrated in Bangui tested positive for human adenoviruses.
The developed method has proved to be useful when it was evaluated by wash teams of Oxfam Intermon in Bangui, Central African Republic and by another team in Pedernales, Ecuador. VirWaTest may be part of an early warning system or may be used in outbreak investigation by organizations involved in water sanitation. This equipment facilitates the analysis of viral pathogens in water and the improvement of water safety management with the objective of reducing incidents of viral diseases in humanitarian crisis contexts.
